Biology Reference
In-Depth Information
Filter with 0.45
m vacuum filtration unit.
Transfer to plastic spray bottle.
m
1 N HCl:
918 mL purified water.
82 mL 37% HCl (add acid slowly to water!).
Filter with 0.2
m nylon filter.
m
15.7.1.3
Procedure
Fill the low volume, 384-well glass-bottom microplate with 20
L DMSO
m
per well.
In a chemical fume hood, set up an ultrasonic water bath with a surface large
enough to fit a microplate (e.g., 15
15 cm).
Carefully place the plate (without lid) on the water surface of the ultrasonic
water bath.
Turn on the sonicator for 10 min. A fine mist of DMSO will appear above the
plate. Do not cover the bath with a lid, as DMSO might be corrosive to it. After
sonication, wipe the bath with a wet paper towel to remove any DMSO that might
have settled from the mist.
Remove the microplate from the surface.
Over a sink, shake off the DMSO from the wells.
Spray the plate evenly with 70% ethanol to wash the wells. Shake off the
liquid into the sink and repeat this ethanol rinse three more times.
Place a regular 384-well plate upside-down on the bench.
Add two sheets of Whatman filter paper cut in 11
7.5 cm pieces.
Place the ethanol washed plate upside-down onto the filter papers.
Load this sandwich in a microplate swinging bucket and balance the rotor.
Centrifuge 5 min at 500
g
.
Inspect a representative sample of the wells using a microscope. These
glass-bottom microplates have variable amounts of glue residues in the wells that
sometimes require several rounds of sonication with DMSO to obtain clean
wells. Repeat the earlier steps up to three times.
Fill the plate with 20
L 1 N HCl per well.
Incubate at RT for 30 min.
Empty wells by shaking off the acid into the sink. (Use appropriate personal
protective equipment, especially goggles!)
Spray the plate evenly with 70% ethanol to wash the wells. Shake off the liquid
into the sink and repeat this ethanol rinse three more times.
Assemble stack with filter papers as the preceding text and centrifuge plate.
Remove plate from stack and cover it with its lid.
The cleaned plate may be stored in a sealed propylene bag for up to 30 days or
used immediately for the next step.
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